Method for quantitative analysis of nonsense-mediated mRNA decay at the single cell level

Nonsense-mediated mRNA decay (NMD) is a ubiquitous mechanism of degradation of transcripts with a premature termination codon. NMD eliminates aberrant mRNA species derived from sources of genetic variation such as gene mutations, alternative splicing and DNA rearrangements in immune cells. In additi...

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Bibliographic Details
Published in:Scientific reports 2015-01, Vol.5 (1), p.7729-7729, Article 7729
Main Authors: Pereverzev, Anton P., Gurskaya, Nadya G., Ermakova, Galina V., Kudryavtseva, Elena I., Markina, Nadezhda M., Kotlobay, Alexey A., Lukyanov, Sergey A., Zaraisky, Andrey G., Lukyanov, Konstantin A.
Format: Article
Language:English
Subjects:
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3' Untranslated regions
3' Untranslated Regions - genetics
631/1647/1888/2249
631/1647/245/2225
631/337/1645/2020
Alternative splicing
Animals
Cell lines
Decay
Deoxyribonucleic acid
DNA
Embryo, Nonmammalian - metabolism
Embryos
Flow Cytometry
Fluorescence
Fluorescence microscopy
Gene expression
Gene regulation
Genes, Reporter
Genetic diversity
Genetic Vectors - metabolism
Green Fluorescent Proteins - metabolism
HEK293 Cells
HeLa Cells
Humanities and Social Sciences
Humans
Mice
Microscopy, Fluorescence
mRNA turnover
multidisciplinary
Nonsense Mediated mRNA Decay - genetics
Nonsense mutation
Nonsense-mediated mRNA decay
Quantitative analysis
RNA Splicing - genetics
Science
Single-Cell Analysis - methods
Xenopus laevis
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Summary:Nonsense-mediated mRNA decay (NMD) is a ubiquitous mechanism of degradation of transcripts with a premature termination codon. NMD eliminates aberrant mRNA species derived from sources of genetic variation such as gene mutations, alternative splicing and DNA rearrangements in immune cells. In addition, recent data suggest that NMD is an important mechanism of global gene expression regulation. Here, we describe new reporters to quantify NMD activity at the single cell level using fluorescent proteins of two colors: green TagGFP2 and far-red Katushka. TagGFP2 was encoded by mRNA targeted to either the splicing-dependent or the long 3'UTR-dependent NMD pathway. Katushka was used as an expression level control. Comparison of the fluorescence intensities of cells expressing these reporters and cells expressing TagGFP2 and Katushka from corresponding control NMD-independent vectors allowed for the assessment of NMD activity at the single cell level using fluorescence microscopy and flow cytometry. The proposed reporter system was successfully tested in several mammalian cell lines and in transgenic Xenopus embryos.
ISSN:2045-2322
2045-2322
DOI:10.1038/srep07729